Role Of Phosphorylation In Tp Receptor Activation

The basal level of TP receptor phosphorylation may influence its activation. Since the previously discussed isomerization states of GPCRs influence ligand binding (See IV. TP RECEPTOR FUNCTION, C. SIGNAL TRANSDUCTION, 4. ROLE OF G PROTEINS above), receptor agonist binding affinity may be influenced by the basal state of phosphorylation. Evidence in favor of this hypothesis comes from the studies of Kinsella, et al and Habib, et al (60,66). Inhibition of PKC by staurosporin resulted in a modest increase in antagonist binding to TP receptors (66), and PKC inhibitors reduced TP receptor basal phosphorylation (60). A PKA inhibitor also increased antagonist binding to TP receptors (66).

PKA and PKC ecto-kinases could contribute to control of TP receptor activation by phosphorylation of the third extracellular loop (66,86, 163). Evidence in support of these kinases having an effect on TP receptor function includes the total inhibition of I-BOP-induced platelet aggregation and of arachidonic acid, EP171 (a stable endoperoxide analog) and U46619-induced platelet aggregation and secretion by alkaline phosphatase that was reversed by pretreatment with inorganic phosphate or ATP plus creatine phosphate/creatine phosphokinase (86,164). Also, studies of a chimeric P7 adrenergic receptor indicated that the third extracellular loop of this GPCR modulated receptor-G protein affinity (153).

Other studies suggested a role for platelet ecto-protein kinase C (ecto-PKC) in inhibition of platelet aggregation via phosphorylation of surface membrane proteins (165). Inhibition of ecto-PKC activity by antibody or inhibitory peptides resulted in spontaneous human platelet aggregation. Platelet aggregation induced by these agents could be blocked by phosphatase inhibitors. Therefore, inhibition of phosphorylation combined with continuous dephosphorylation, that resulted in a decrease in the phosphorylation state of the membrane, appeared to be responsible for platelet aggregation. No evidence of effects on receptor phosphorylation was presented, but the anti-ecto-PKC antibody inhibited phosphorylation of an ~50 kDa membrane protein. The third extracellular loop of the TP receptor is in a position where it could be phosphorylated by ecto-PKC. However, evidence against an effect of phosphorylation on TP receptor binding is the observation that neither the affinity nor the number of [125I]-BOP platelet binding sites was altered by pretreatment with alkaline phosphatase (164). Alkaline phosphatase significantly decreased I-BOP-stimulated GTPase activity, and addition of inorganic phosphate to platelets exposed to alkaline phosphatase and I-BOP resulted in aggregation within seconds, plus restoration of increased I-BOP-induced GTPase activity. These studies indicated that the phosphor-ylation state of TP receptors, or closely associated proteins, influenced TP receptor-mediated platelet activation, but the mechanism responsible was not defined.

In addition to the serine-threonine phosphorylation sites modulated by PKC and PKA, a basal state of tyrosine phosphorylation exists in platelet TP receptors, as in bradykinin receptors, and this phosphorylation increases on agonist stimulation (166). The tyrosine kinase responsible for this phosphorylation, as well as that of the parallel phosphorylation of phosphatidylinositol 34rinase (PI3-kinase), that occurs on agonist stimulation, is unknown, but p27,1'lt• known to be activated by TP receptor agonists (167), is a candidate (166). It is also possible that PI} kinase itself might phosphorylate TP receptors. The functional role of tyrosine kinase-mediated phosphorylation of TP receptors is unknown.